Tick-borne pathogens in questing adults Dermacentor reticulatus from the Eastern European population (north-eastern Poland)

Dermacentor reticulatus is tick species with an expanding geographical range in Europe, which creates the possibility of spreading microorganisms of significant veterinary and medical importance. The study aimed to investigate the prevalence and genetic diversity of Rickettsia spp., Babesia spp., Borrelia spp. and Anaplasma phagocytophilum in adult D. reticulatus ticks from the Eastern European population in the urban and the natural biotopes of north-eastern Poland. Microorganisms were detected by PCR and identified by DNA sequencing. The overall infection rate of at least one of the pathogens was 29.6%. The predominantly was Rickettsia spp. (27.1%) (with R. raoultii—9.1%) followed by Babesia spp. (2.4%) with B. canis (1.5%) as the most frequent. Based on 18S rRNA gene sequence, three B. canis genotypes were revealed. The prevalence of R. raoultii and B. canis was significantly higher in ticks from natural biotopes. The infection rates of B. afzelii and A. phagocytophilum were determined at 0.9% and 0.3%, respectively. Co-infections were detected in 3.8% of infected ticks. In diagnosing tick-borne diseases in humans, tick-borne lymphadenopathy should not be excluded. The prevalence of different genotypes of B. canis suggests differences in the clinical picture of canine babesiosis in the area.

Dermacentor reticulatus is the second most abundant tick species after Ixodes ricinus in central Europe 1,2 .Until the 1980s, the geographical range of D. reticulatus was clearly divided into two main zones: the Western European and the Eastern European populations.In between, there was a large area from the Baltic Sea coast through central Germany, western Poland to the southern border of Hungary where the meadow tick had never been observed 3,4 .Currently, those two populations of D. reticulatus (Western and Eastern) are among the most dynamic tick populations in Central Europe 5 .In several European countries (Slovakia, the Czech Republic, the United Kingdom, the Netherlands, and Germany), habitat expansion of D. reticulatus was noted 6 , and the northern distribution border in the Baltic countries (Lithuania, Latvia) moved further to the north 7 .On the Polish territory in "the gap" zone, new foci occurrences of this tick species have also been reported 5,[8][9][10] which leads to closer borders between the western and the eastern D. reticulatus populations.
With the expansion of D. reticulatus into new areas, the veterinary-medical importance of this species has increased due to the variety of transmitted pathogenic microorganisms 11 .D. reticulatus is considered the main vector of the protozoa Babesia canis (the etiological agent of canine babesiosis), as well bacteria of the genera Rickettsia, Anaplasma, Bartonella, Francisella, Coxiella burnetii 2,12 and tick-borne encephalitis virus 13 .The significance of D. reticulatus in the transmission of Borrelia burgdorferi s.l. is still unclear 2,12 .Not without significance is also the contribution of D. reticulatus ticks to the maintenance of tick-borne pathogens in the environment through the transfer of pathogenic microorganisms between various vertebrate hosts, which are susceptible to infection and serve as efficient reservoirs 12 .
The eastern population of D. reticulatus ticks in Poland is considered a stable population and is the source of newly emerging foci of this species in the zone previously considered free (central and western Poland) 5,9 , and

DNA extraction
The genomic DNA from D. reticulatus ticks was extracted using the ammonia method 16 .Before DNA extraction, ticks preserved in 70% ethanol were air-dried and then separately cut and crushed using a sterile mortar in 0.7 M ammonium hydroxide (NH 4 OH).The obtained DNA lysates were stored at − 20 °C for further molecular analysis.

Pathogen DNA detection
Tick DNA samples were used to detect tick-borne microorganisms.For the detection of DNA of Babesia spp., a nested PCR (nPCR) reaction targeting the 18S rRNA gene using the primers CRYPTO F/CRYPTO R and Bab GF2/Bab GR2 [17][18][19] was used (Table 1).The presence of Rickettsia spp. in tick genomic DNA samples was confirmed by using a set of primers (CS409/Rp1258) 20 specific to the fragment of the citrate synthase (gltA) gene (Table 1). A. phagocytophilum DNA was detected using the nPCR with two sets of primers (ge3a/ge10r and ge9f/ ge2) targeting fragments of the 16S rRNA gene 21 (Table 1).Detection of B. burgdorferi s.l.DNA was performed via the nPCR-restriction fragment length polymorphism (RFLP) method using primers 132f/905r and 202f/832r specific to the flaB gene 22,23 (Table 1).The identification of species belonging to the Borrelia burgdorferi s. l. complex was based on the restriction patterns obtained by digestion of the inner-PCR product (604 bp) by using the restriction enzyme HpyF3I (DdeI) (ThermoFisher Scientific, Waltham, MA, USA) 22,23 .
All amplifications were performed in a total volume of 25 µL of PCR mixture containing 12.5 µL of 2× PCR Master Mix Plus (0.1 U/µL of Taq polymerase supplied in a PCR buffer, 4 mM of MgCl 2 and 0.5 mM of each dNTPs) (A&A Biotechnology, Gdynia, Poland), 0.5 µL of each primer (10 µM), 2-4 µL of template DNA (in nPCR-1 µL of template DNA or 1 µL of the outer-PCR product) and an appropriate amount of sterile nucleasefree water.DNA of B. canis isolated from the blood of an infected dog, Rickettsia spp., A. phagocytophilum and B. afzelii obtained from an infected I. ricinus tick (confirmed by sequencing in earlier studies) were used as a

Identification of pathogen species
To confirm the species of the detected microorganisms, selected PCR products (81 for Rickettsia spp., 21 for Babesia spp., three for A. phagocytophilum, five for Borrelia spp.) were purified using the CleanUp purification kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol and sequenced bi-directionally (Macrogen Europe, Amsterdam, the Netherlands) with forward and reverse primers.The obtained nucleotide sequences were edited in BioEdit v. 7.2 software (https:// bioed it.softw are.infor mer.com, accessed on February 2022) and compared with data registered in the GenBank database (http:// www.ncbi.nih.gov/ Genba nk/ index.html, accessed on May 2023) using the BLAST-NCBI program (http:// www.ncbi.nlm.nih.gov/ BLAST).
Consensus sequences were deposited in the GenBank database and registered under the accession numbers ON660868-870 for the gltA gene of Rickettsia spp., OR056353, OR064513-518 for the 18S rDNA of Babesia spp., OR096226-227 for the 16S rDNA of A. phagocytophilium and OR046059-060 for the flaB gene of Borrelia spp.
The phylogram was constructed using the Maximum Likelihood method based on the Kimura 2-parameter model.The topology of the phylogenetic tree was evaluated using the bootstrap method with 1000 replicates.The phylogenetic analysis was conducted using MEGA X software (https:// www.megas oftwa re.net).

Statistical analysis
A chi-square test (with post-hoc Bonferroni test) and 95% confidence intervals (95% CI) were used to compare differences in the prevalence of detected microorganisms in the sex of ticks, regions, habitats and years of study.In all analyses, p-values below 0.05 were considered statistically significant.The analysis was conducted using the software package SPSS version 27.0 for Windows (SPSS Inc., Chicago, USA).
All A. phagocythophilum-positive samples were confirmed by nucleotide sequencing.All of them were identical and showed 100% similarity to the sequence of A. phagocythophilum derived from the blood of red deer in Czechia (GenBank: EU839849) and of Merino sheep in Germany (GenBank: MZ348273).Borrelia spp. in D. reticulatus samples were identified as B. afzelii.The sequence showed 100% identity to the strains BO23 (Gen-Bank: CP018262) and K78 (GenBank: CP009058) that were detected in symptomatic patients with borreliosis in Germany and Austria.

Discussion
The authors' previous monitoring of tick occurrence in north-eastern Poland demonstrated that D. reticulatus is permanently present in both natural biotopes as well in the green areas located in cities [24][25][26][27] .The present study focused on molecular evidence of veterinary-medical important pathogens transmitted by questing D. reticulatus ticks belonging to the Eastern European population from urban and natural biotopes of a region of north-eastern Poland.The presence of genetic material of all four studied groups of microorganisms (Rickettsia spp., Babesia spp., A. phagocytophilum, Borrelia spp.) was found in the study, and almost every third D. reticulatus tick (29.6%) was infected with at least one of them, regardless of sex.
From an epidemiological point of view, D. reticulatus ticks are less important than I. ricinus ticks.In veterinary practice, the most important pathogen transmitted by D. reticulatus is B. canis protozoa, which causes canine babesiosis 28 .In the current study, B. canis was identified in D. reticulatus ticks in similar proportions in both urban and natural biotopes.However, the overall level of infection was relatively low (1.4%) compared to ticks from other sites in the eastern macroregion.The prevalence of B. canis in ticks from the Eastern European population has been reported to vary from 0 to 6.8% 19,25,[29][30][31][32][33][34] .It is worth underlining that in the current study, most of the D. reticulatus infected by B. canis were detected in an urban area of the city of Olsztyn in the central part of the Warmia and Mazury region, the oldest area known as endemic for D. reticulatus 3,5 .These results confirm the presence and relatively constant level of prevalence B. canis (2.5%) in this part of north-eastern Poland, previously established by other authors in the range of 2.3-6% 5,19 .
In spite of a low overall prevalence, the molecular analysis of a fragment of the 18S rRNA gene of B. canis indicates the presence of genetically heterogenic genotypes of B. canis in examined ticks.In Europe, based on two single nucleotide polymorphisms (SNPs), four B. canis genotypes related to GA → AG nucleotide substitutions are present at different rates of prevalence [35][36][37] .In the present study, among three revealed genotypes, the vast majority of B. canis isolates (69.2%) represented genotype A (GA nucleotides).However, the "mixed" A/B genotype (showing the presence of both G and A nucleotides-R/R) has also been recorded.The A/B genotype was the most frequently detected in naturally infected dogs in Poland and other countries from northern Europe 37,38  In 0.9% of examined D. reticulatus ticks, the DNA of B. microti, considered to be the most common causative agent for human babesiosis, was also detected 40 .Interestingly, it was detected only in females from urban areas.Although the occurrence of DNA of B. microti was previously reported in D. reticulatus 19,25,30,41 , a role for this tick species as a vector for B. microti has not been clearly confirmed.The presence of B. microti DNA in questing D. reticulatus ticks may be due to feeding on voles (Microtus spp.), which have been identified as the main reservoir of B. microti 42 .
"Contamination" with the blood of the host is probably also the cause of the detection of Borrelia spp.DNA in examined D. reticulatus ticks.Although the specific DNA of that bacteria has been detected in D. reticulatus in other regions of Poland 19,26,27,30 , their role as a vector was not confirmed.It has been proven that the salivary glands of Dermacentor spp.contain proteins (defensins), which are attributed to the role of specific antibiotics in eliminating spirochetes 43 .
The significance of D. reticulatus as a vector of A. phagocytophlium-the causative agent of human granulocytic anaplasmosis (HGA) 44 also seems to be insignificant.A meta-analysis of the prevalence and distribution of A. phagocytophilum in tick vectors conducted by Karshim 45 showed that the overall level of infection with this pathogen in questing D. reticulatus ticks is very low (0.42%).The current results (0.3%) correspond with the results of other studies on questing D. reticulatus ticks belonging to the East European population (0.7-3%) 30,33,[46][47][48] and confirmed that fact.However, it should be noted that the infection rate of A. phagocytophilum in D. reticulatus may vary significantly depending on the year of study, the local availability of hosts and the phase of the life cycle (non-feeding/feeding on the host) 26,27,30,33,34,[46][47][48][49][50][51] .
From a medical point of view, D. reticulatus is of undeniable importance in the transmission of bacteria representing the genus Rickettsia 12 .Although D. reticulatus ticks attack humans sporadically [52][53][54] , it is a main vector of R. raoultii and R. slovaca.Tick-borne lymphadenopathy (TIBOLA)/Dermacentor-borne necrosis erythema-lymphadenopathy (DEBONEL) or scalp eschar and neck lymphadenopathy after a tick bite (SENLAT) are recently described infection syndromes in humans caused by R. raoultii and R. slovaca belonging to spotted fever group (SFG) rickettsiae 55,56 .To date, several cases of DEBONEL/TIBOLA have been described in Europe, including in Poland [57][58][59][60][61] .R. raoultii is the most commonly detected pathogen in both adult and juvenile D. reticulatus ticks in Poland 62 .R. raoultii was the only species identified by DNA sequencing in 33% of Rickettsiapositive ticks in this study.The frequency of this pathogen in the examined specimens ranged from 6.3 to 21.8% depending on the research region and was much higher in ticks collected from natural biotopes than in urban areas (the city of Olsztyn).The level of occurrence of R. raoultii in ticks in north-eastern Poland was comparable to that previously determined by Mierzejewska 19 in this area (34.2%).A higher infection rate, ranging from 41 to 91.7%, was detected in adult D. reticulatus in populations in other parts of north-eastern and eastern Poland 34,[63][64][65] .Mierzejewska 19 , comparing the prevalence of R. raouliti between two Polish tick populations (eastern and western) with an expansion zone between them, noted slight differences (42% in the East vs. 52% in the West), but with a clearly increasing gradient from east to west in Poland.A high prevalence of Rickettsia spp. in the population of D. reticulatus ticks may result from the possibility of transmission not only through the infected host-tick route, but also via the vertical transovarial, transstadial and, probably less frequently, transspermal transmission 56 .
In the case of tick-borne diseases in humans, tick co-infection with several species of pathogenic microorganisms and their co-transmission might have important relevance to public health 66 .In D. reticulatus from north-eastern Poland, co-infections were revealed in 3.8% of positive ticks.Considering that only co-infections with R. raoultii with B. canis were identified (which are not pathogenic for humans) and with B. afzelii (which are probably neutralized by defensins produced by D. reticulatus ticks), it seems that this tick species does not play an important role as a vector of mixed tick-borne infections in humans that could exacerbate the course of the disease severity and make it difficult to diagnose or treat.

Conclusion
The current study has confirmed the high prevalence of R. raoultii in adult ticks in the eastern population of D. reticulatus in north-eastern Poland.The presence of bacteria belonging to the rickettsiae from the spotted fever group indicates that tick-borne lymphadenopathy (DEBONEL/TIBOLA) should not be excluded in the diagnosis of tick-borne diseases.In turn, the relatively high prevalence of B. canis with different genotypes suggests differences in the clinical picture of canine babesiosis in this area.The risk of infection with these two pathogens is much higher in the natural biotopes of north-eastern Poland.D. reticulatus seems to play a minor role in the transmission of Borrelia burgdorferi s.l. and A. phagocytophilum, as well as a vector of mixed infections in humans.

Figure 1 .
Figure 1.Dermacentor reticulatus tick collection sites located in north-eastern Poland.The map was designed in CorelDRAWX5 based on Google Maps (https:// www.google.pl/ maps).

Figure 2 .
Figure 2. Phylogenetic relationships between Rickettsia raoultii identified in the study and accessions from GenBank, based on the sequences of the gltA gene of Rickettsia spp.The phylogram was constructed conducted in MEGA X software (https:// www.megas oftwa re.net) using the Maximum Likelihood method and the Kimura 2-parameter method as a distance method.The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1000 replicates) is shown next to the branches.The tree is drawn to scale, with branch lengths measured in the number of base substitutions per site.The sequences obtained in this study are labelled with black symbols.

Table 2 .
Total infection rate in Dermacentor reticulatus ticks by sex, year, region and biotope in northeastern Poland.*χ 2 test, p < 0.05.a,b Different letters mean significant differences (post-hoc Bonferroni test).

Table 4 .
. The occurrence of genotype A and genotype B (AG nucleotides) of B. canis in D. reticulatus in north-eastern Poland may suggest differences in the clinical manifestation of canine babesiosis in this area.Adaszek 39 revealed Co-infection with the pathogens of the genera Rickettsia, Babesia, Anaplasma and Borrelia in Dermacentor reticulatus ticks from north-eastern Poland.F female, M male.www.nature.com/scientificreports/ that the severity of thrombocytopenia in dogs infected with B. canis is related to different 18S rRNA genotypes of the pathogen.Genotype B of B. canis, also identified in the current study, was found to be more virulent in relation to thrombocytopenia than genotype A.